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研究生:陳建軒
研究生(外文):Chien-HsuanChen
論文名稱:阿拉伯芥鹼性半乳糖苷酶蛋白質家族功能性分析
論文名稱(外文):Functional Study of Alkaline α-Galactosidase Protein Family in Arabidopsis
指導教授:黃浩仁
指導教授(外文):Hao-Jen Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:熱帶植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:85
中文關鍵詞:阿拉伯芥鹼性半乳糖苷酶荷爾蒙非生物逆境基因表現功能分析
外文關鍵詞:Arabidopsisalkaline a-galactosidasehormonesabiotic stressesexpression profiles
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葉片老化(leaf senescence)是基因調控葉器官生長發育的最後程序,受到相當複雜的機制所調控,屬於一種計劃性細胞死亡,使有利用價值的營養能分解並重新分配到植株其他需要的部位再利用。葉片老化受到內在因子(如年齡、生殖生長和植物荷爾蒙等)以及外在環境因子(如極端溫度變化、空氣汙染物、缺乏養份或水分、病菌感染和機械傷害等)所影響。鹼性半乳糖苷酶是在植物特有酵素,只在中、鹼性環境下才具有活性,主要作用的對象是含有非還原端和側鏈帶有α-D galactosyl殘基的醣類如raffinose family of oligosaccharide(RFO)和醣脂digalactosyl diacylglycerol (DGDG)等受質,這些受質在植物的生長發育及抗非生物逆境上扮演重要角色。阿拉伯芥基因體中一共有3個鹼性半乳糖苷酶基因(At1g55740,At3g57520,At5g20250)位於第1,3,5染色體上,為了解這些基因如何受荷爾蒙及不同非生物逆境調控,本研究以荷爾蒙及非生物逆境做處理並利用RT-PCR觀察鹼性半乳糖苷酶基因表現模式,發現會明顯受離酸、茉莉酸等荷爾蒙以及冷、熱、滲透壓等逆境影響而有不同表現模式。此外也利用分離阿拉伯芥的原生質體進行短暫表現分析,觀察到不同鹼性半乳糖苷酶和報導基因GFP的融合蛋白會因RNA轉錄本剪接型式的不同而改變胞器表現位置。我們也建構並分離大量基因表現轉植株和T-DNA knockout突變株,同時也建構病毒誘導基因靜默(VIGS)載體,希望這些基因表現異常的植株及短暫基因靜默法能用來輔助探討不同鹼性半乳糖苷酶在植物生長發育及不同逆境下所扮演的基因功能。
Leaf senescence is a genetically programmed process that ultimately leads to organ death. It involves complex and highly regulated molecular and cellular events in response to developmental and environmental signals that allow efficient recycling of nutrients to other sink tissues. Alkaline a-galactosidases hydrolyze a variety of glycosides possessing non-reducing terminal and side-chain a-galactosyl residues such like raffinose family of oligosaccharide (RFO) and digalactosyl diacylglycerol (DGDG). These substrates are essential for plant growth and development, and against unfavorable environment. There are three alkaline a-galactosidases in Arabidopsis genome (At1g55740, At3g57520, At5g20250). We are interested in understand the physiological function of the protein family using Arabidopsis as model plant. In this study, the expression profiles of alkaline a-galactosidase transcripts in Arabidopsis seedings were examined under different phytohormones and abiotic stress treatments. Our results show ABA, JA, extreme temperature and osmotic stress significantly affect the expression levels of these genes. Arabidopsis alkaline a-galactosidase-GFP fusion products also show that they can express in different subcellular locations on different splicing variants. Arabidopsis overexpression transgenetic plants and T-DNA insertion mutants for these genes were isolated, and these plants ectopic expressed these alkaline a-galactosidases genes together with VIGS vector constructs will be use for functional studies of these genes in response to developmental and environmental signals.
緒論...............................................................................1
一. 調控葉片老化內外在因素………………………………………..2
1. 內在調控因子…………………………………………………..2
1.1 植物荷爾蒙………………………………………………..2
1.1.1 乙烯(Ethylene)…………………………………….2
1.1.2 茉莉花酸(Jasmonic acid)………………………..3
1.1.3 水楊酸(Salicyclic acid)…………………………..4
1.1.4 離層酸(Abscisic acid)…………………………….5
1.1.5 細胞分裂素(Cytokinin)…………………………..5
1.1.6 生長素(Auxin)……………………………………...8
1.1.7 吉貝素(Gebberrllin)………………………………9
1.2 糖…………………………………………………………….9
2. 外在調控因子…………………………………………………10
2.1 溫度的變化……………………………………….………10
2.2 水分及養分……………………………………….………11
2.3 空氣污染………………………………………………….11
2.4 病蟲害…………………………………………………….11
2.5 光…………………………………………………….…….12
二. 鹼性半乳糖苷酶蛋白質族群在調控葉片老化及抗非生物逆境其生理功能………………………………………………………….12
實驗目的..........................................................................15
材料與方法...................................................................16
1. 阿拉伯芥幼苗的培養與處理………………………………….16
2. 阿拉伯芥盆栽方法…………………………………………..…16
3. 蛋白質序列演化樹分析……………………………….……...17
4. 啟動子序列分析………………………………………………...17
5. 預測植物蛋白質在細胞內表現位置…………………………17
6. 細菌培養基配製………………………………………………...17
7. 接合作用…………………………………………………………18
8. 限制酵素切割作用……………………………………………..18
9. 大腸桿菌的轉型作用…………………………………………..18
10. TA克隆………………………………………………………..19
11. 藍白菌落篩選…………………………………………………19
12. 分離質體DNA………………………………………………..19
13. 阿拉伯芥葉片原生質體(Arabidopsis mesophyll protoplasts)製備………………………………………………..20
14. PEG (polyethylene glycol)轉染法…………………………21
15. 農桿菌之重組質體轉形……………………………………..21
16. 轉殖阿拉伯芥…………………………………………………22
17. 篩選與種植阿拉伯芥………………………………………..22
18. 總量RNA (total RNA) 之萃取…………………………..23
19. 反轉錄聚合酶鏈鎖反應(RT-PCR, Improm IITM Reverse Transcriptase, Promega,USA)………………………………23
20. 聚合酶鏈鎖反應( PCR,GoTaq Flexi DNA Polymerase, Promega, USA )………………………………………………..24
實驗結果.......................................................................26
1. 利用啟動子序列分析預測阿拉伯芥幼苗的鹼性半乳糖苷酶受荷爾蒙及逆境影響表現情形…….……………………26
2. 阿拉伯芥幼苗的鹼性半乳糖苷酶受不同荷爾蒙調控基因表現模式………………………………………………………26
3. 阿拉伯芥幼苗的鹼性半乳糖苷酶受不同非生物逆境調控基因表現模式………………………………………………..27
4. 阿拉伯芥ABA突變株的生長情形以及鹼性半乳糖苷酶RNA表現………………………………………………………27
5. 構築大量表現載體………………………………........…….28
6. 蛋白質細胞表現位置分析……………………………………28
7. 阿拉伯芥轉殖及篩選…………………………………………29
8. 構築病毒誘導基因靜默載體………………………………..29
討論.............................................................................30
結論..................................................................................33
參考文獻.........................................................................34
表...................................................................................42
圖...................................................................................53
附圖...............................................................................83

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